G protein-coupled receptors (GPCRs) mediate hormonal control of numerous signaling pathways, many of which are dynamically regulated. At the level of the receptor, regulation can occur via inhibition of GPCR/G protein coupling (desensitization), redistribution of cell surface receptors (trafficking), or receptor degradation (down-regulation). Two protein families, GPCR kinases (GRKs) and arrestins, play a critical role in these processes. GRKs specifically phosphorylate the activated form of the receptor, which in turn promotes arrestin binding. Arrestin interaction has been directly linked to many processes including GPCR desensitization, trafficking, and G protein-independent signaling. In the initial period ofthe MERIT award, we focused on characterizing the role of arrestin interaction with other proteins and how such interactions mediate the biological effects of arrestins. In addition, we used biophysical approaches to better understand how arrestins mediate receptor trafficking. These studies have resulted in 14 peer-reviewed publications as well as 5 manuscripts in various stages of submission. In the MERIT award extension period, we propose to continue our work in four areas. The first will involve further characterizing the functional role of arrestin interactions with a number of target proteins that we have already identified including RCC2, API and PTEN. The second area involves correlating the functional and biological roles of arrestin interactions in cell lines and in C. elegans. A third area will involve the use of biophysical approaches to better understand the scaffolding properties of arrestins and how receptor binding regulates arrestin conformation and interaction. A final area that we plan to pursue involves characterizing the link between arrestins and two other protein families (Vps26 and a-arrestins) that appear to have structural similarities with the arrestins. Overall, our efforts will provide unique mechanistic insight into the biochemical, cellular and molecular function of arrestins and should prove important in understanding diseases where GPCR signaling defects are observed. RELEVANCE (See instructions): Arrestins have a broad biological role in various organisms although relatively little is known about how the function of arrestins in cells correlates with the observed biology. Our proposed research will provide unique mechanistic insight into the biochemical, cellular and molecular function of arrestins and should prove important in understanding the biological function of arrestins and their role in disease.